X chromosome inactivation is an extraordinary example of long-range gene regulation, extending ~150 megabases and silencing genes on one X in females as a means of equalizing gene dosage between XX females and XY males. Nonetheless, not all genes on the X are silenced. Mechanistically, how inactivation spreads along the X and why some regions "escape" X inactivation are not well understood but are important questions. Indeed, few examples of gene regulation are so intimately tied to chromosome organization, evolution and disease. Current data support a role for underlying genomic sequence and insulation by boundary elements. Human escape genes are not rare but largely cluster, suggesting that they are organized in coordinately controlled domains. We will use comparative genomics and molecular genetics tools to address the following hypotheses: (1) Underlying genomic sequences and boundary elements regulate X inactivation and are evolutionarily conserved. This will be tested by determining X inactivation patterns in ten mammals and developing a computational and statistical platform to identify candidate regulatory sequences, (2) Insulators are a conserved mechanism to regulate escape genes. This will be tested by characterizing epigenetic features of a human insulator that lies in an escape transition and determining whether insulator function correlates with escape domains in other mammals, (3) Genomic landscape functionally influences escape gene expression, and (4) Escape gene mechanisms are functionally conserved. To address these last two hypotheses, we will introduce mouse and human escape genes to different locations on the mouse X and and determine whether this affects their expression on the inactive X. The proposed experiments have direct relevance for medical genetics. The inheritance of an abnormal number of X chromosomes is quite common, accounting for 1 in 650 live births. One specific case, Turner syndrome, is the most common genetic birth defect in females. Many problems in these individuals are due to the specific subset of genes that will be studied in this application. We need to better understand these genes to explain clinical features and to improve genetic counseling recommendations. Further, these studies will also give insight into why genes are silenced when they are placed into new chromosomal environments, such as chromosome rearrangements that commonly occur in cancers and gene insertions for gene therapy.